Document Detail


G0-G1 cell cycle phase transition as revealed by fluorescence resonance energy transfer: analysis of human fibroblast chromatin.
MedLine Citation:
PMID:  15145774     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In the present study, microspectrofluorometry and digital imaging procedures were used to investigate by fluorescence Resonance Energy Transfer (FRET) analysis the changes of chromatin organization during the transition from G0 quiescent stat to G1 phase. G0 transition is a key event in cell cycle progress depending on the activation of specific genes and the concomitant silencing of others, which both entail spatial chromatin rearrangement. Normal human fibroblasts arrested in G0-phase by culture in low-serum containing medium and stimulated to re-enter G1 by serum addition were used as cell model. To investigate the occurrence and timing of these supramolecular chromatin changes, we estimated the relative FRET efficiency in single cells after double-helical DNA. Hoechst 33258 amd propidium iodide were used as a donor-acceptor dye pair since they exhibit particularly favourable spectral characteristics, that allow the calculation procedure to be semplified. The results of FRET analysis were compared to those of the immunocytochemical labelling of two nuclear proteins (i.e., Ki-67 and statin) whose expression is an established marker of potentially proliferating G1 cells or resting G0 cells, respectively. FRET efficiency was lower in G0 than G1 fibroblasts: this is likely due to higher chromatin packaging in quiescent cells which especially hinders the interaction with the donor molecules less favourable, in terms of relative distance and spatial orientation. FRET efficiency significantly increased shortly (1h) after serum stimulation of quiescent fibroblasts, thus indicating that chromatin is rearranged in parallel with activation of cycle-related gene; it is worth noting that these signs largely preceded the occurrence of immunopositivity for Ki-67, which was detectable only 24h after serum stimulation. FRET-based analyses which already proved to be suitable for studying the overall chromatin organization in differentiated cells, may now be envisaged as a powerful tool for detecting, in single cells, more subtle changes linked to the activation of early cycle-related genes.
Authors:
Giovanni Bottiroli; A C Croce; M G Bottone; S Vaccino; C Pellicciari
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  European journal of histochemistry : EJH     Volume:  48     ISSN:  1121-760X     ISO Abbreviation:  Eur J Histochem     Publication Date:  2004  
Date Detail:
Created Date:  2004-05-17     Completed Date:  2004-11-09     Revised Date:  2008-08-15    
Medline Journal Info:
Nlm Unique ID:  9207930     Medline TA:  Eur J Histochem     Country:  Italy    
Other Details:
Languages:  eng     Pagination:  37-48     Citation Subset:  IM    
Affiliation:
Istituto di Genetica Molecolare del CNR, Sezione di Istochimica e Citometria, piazza Botta 10, 27100 Pavia, Italy. botti@igm.cnr.it
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MeSH Terms
Descriptor/Qualifier:
Binding Sites
Cell Cycle / physiology*
Cells, Cultured
Chromatin / chemistry*,  metabolism
Cytokines / metabolism
DNA / metabolism
Fibroblasts / chemistry*,  cytology,  metabolism
Fluorescence Resonance Energy Transfer / methods*
G0 Phase
G1 Phase
Humans
Spectrometry, Fluorescence / methods
Staining and Labeling
Chemical
Reg. No./Substance:
0/Chromatin; 0/Cytokines; 9007-49-2/DNA

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